Endothelin (ET) is an endogenous peptide which has been implicated in numerous physiological and pathological phenomena within the body. Acting upon two distinct receptors, ETA and ETB, ET influences a range of processes from regulation of blood pressure to neurotransmitters and hormones (Kojima et al., 1992; Levin, 1995; Schiffrin et al., 1997; Schneider et al., 2007). Although most widely studied for their actions on cardiovascular system, ET receptors are widespread throughout the body, including the brain. ETB receptors, specifically, are located in abundance on neurons and glial cells, as well as endothelial lining of the cerebral vasculature (Schinelli, 2006). The exact function of these receptors within the brain, particularly during its development, is not well understood.
Development of the Central Nervous System
A deficiency in ETB receptors at birth has been shown to result in a decrease in neuronal progenitor cells and an increase in apoptosis within the postnatal dentate gyms and cerebellum of rats (Ehrenreich et al., 2000; Vidovic et al., 2008). Additionally, ETB knockout model in rats leads to congenital aganglionosis within the gut and associated CNS disturbances (Dembowski et al., 2000). These ETB knockout rats, which have a 4 week postnatal mortality, serve as models for human Hirschsprung disease. Previous studies have shown that brain ETB receptor expression is particularly high immediately after birth, but drops down to lower levels by postnatal day 21 (Briyal et al., 2012b). The locations of these receptors and their correlation, or lack thereof, with CNS growth factors during these crucial stages of development remain to be determined.
While it is clear that ETB receptors are needed for normal CNS development, it remains uncertain which cells or pathways they exert a protective or proliferative influence on. Previous studies have shown that selective stimulation of ETB receptors produces neuroprotection against oxidative stress and a significant reduction in infarct volume in the brains of adult rats subjected to cerebral ischemia (Leonard et al., 2011; 2012). It was also found that protection and recovery from the ischemic condition was at least partially due to an increase in angiogenesis and neurogenesis within 7 days following infarct and treatment with ETB receptor agonist, IRL-1620 (Leonard and Gulati, 2013). An increase in vascular and nerve growth factors within the brain of IRL-1620-treated infarcted animals coincided with an increase in the level of ETB receptors.
Vascular endothelial growth factor (VEGF) is expressed normally in the cerebral microvessels as well as in the neuronal tissue of both neonates and adults (Hoehn et al., 2002). VEGF in the fetal human brain is located on neuroepithelial cells, neuroblasts, radial glial cells and endothelial cells, and its expression appears to be developmentally regulated and correlated with angiogenesis (Virgintino et al., 2003). While VEGF is well known to be necessary for blood vessel growth, recent research has indicated that it also plays a significant role in promoting neurogenesis, neuronal patterning, and neuronal migration (Rosenstein et al., 2010). It has been shown that there is a correlation between VEGF, neuronal growth factor (NGF) and ETB receptors in the developing brain. ETB receptors can be stimulated by administering ETB receptor agonists such as IRL-1620 and growth of the CNS treat diseases can be promoted where CNS has been damaged or has not grown appropriately.
Neurodegenerative Diseases
Neurodegeneration is a term for the progressive loss of structure or function of neurons, including death of neurons. Many neurodegenerative diseases including Amyotrophic lateral sclerosis (ALS), Parkinson's, Alzheimer's, and Huntington's occur as a result of neurodegenerative processes. As research progresses, many similarities appear that relate these diseases to one another on a sub-cellular level. Discovering these similarities offers hope for therapeutic advances that could ameliorate many diseases simultaneously. There are many parallels between different neurodegenerative disorders including atypical protein assemblies as well as induced cell death (Bredesen et al., 2006; Rubinsztein, 2006).
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cerebrovascular and neuronal dysfunctions leading to a progressive decline in cognitive functions. Neuropathological hallmarks of AD include beta amyloid (Aβ) plaques and neurofibrillary tangles (Johnson et al., 2008). It has long been speculated that cerebrovascular dysfunction contributes to AD. Aβ has been shown to decrease myogenic response, cerebral blood flow (CBF) and vasodilator responses (Han et al., 2008; Niwa et al., 2000; Paris et al., 2004; Shin et al., 2007). Regulation of CBF tends to be impaired in transgenic mice with high intracerebral levels of Aβ (Niwa et al., 2002). Synthetic Aβ has been shown to impair endothelin (ET) dependent relaxation and enhance vasoconstriction in vivo and in vitro (Niwa et al., 2000; Niwa et al., 2001).
Several studies have demonstrated an involvement of ET in AD. ET is an endogenous vasoregulatory peptide which targets two main receptors—ETA and ETB. ETA receptors are mainly located on vascular smooth muscle cells and mediate vasoconstriction, whereas ETB receptors are mainly located on vascular endothelial cells and mediate vasodilatation (Goto et al., 1989; Tsukahara et al., 1994). ET has been demonstrated to be present in the brain and plays an important role in the regulation of cerebral and systemic blood circulation (Gulati et al., 1997; Gulati et al., 1996; Gulati et al., 1995; Rebello et al., 1995a). It was initially demonstrated that ET-1 concentrations in the cerebrospinal fluid of patients with AD were lower compared to control (Yoshizawa et al., 1992), however, subsequent studies indicate that ET-1 like immunoreactivity was significantly increased in the cerebral cortex (frontal and occipital lobes) of patients that suffered from AD compared to control brains (Minami et al., 1995). Brain samples of AD patients obtained post mortem showed increased expression of ET-1 immunoreactivity in astrocytes (Zhang et al., 1994). It has been suggested that ET-1 released from astrocytes may reach the vascular smooth muscle cells and induce vasoconstriction. ET binding sites in the human brains with AD were found to be decreased which could be due to loss of neurons in the cortex (Kohzuki et al., 1995).
The mechanism by which soluble Aβ interferes with vascular function is not fully understood. A possible mechanism by which soluble Aβ interferes with vascular function may be mediated through ET-1 which plays a central role in the regulation of cardiovascular functions and regional blood flow (Gulati et al., 1997; Gulati et al., 1996; Gulati et al., 1995). It was previously found that specific ETA receptor antagonists (BMS182874 and BQ123) prevent Aβ induced oxidative stress and cognitive deficits (Briyal et al., 2011). Specific ETA receptor antagonists reduced escape latencies and also increased preference for the target quadrant. On the other hand, a nonspecific ETA/ETB receptor antagonist (TAK-044) did not produce any improvement in spatial memory deficit or loss of preference for the target quadrant (Briyal et al., 2011). This lack of improvement with the non-specific ETA/ETB antagonist indicated to us the specific involvement of ETB receptors in AD.
ET binding sites in the brain are predominantly of ETB receptors, and ETB receptor agonists have been shown to be anti-apoptotic against neurotoxicity of Aβ (Yagami et al., 2002). Complete deficiency or blockade of ETB receptors leads to exacerbation of ischemic brain damage, possibly due to the shift in ET vasomotor balance (Chuquet et al., 2002; Ehrenreich et al., 1999). It has been demonstrated that activation of ETB receptors with intravenous IRL-1620, a highly selective ETB agonist, results in a significant elevation in CBF in normal rats and reduction in neurological deficit and infarct volume of stroked rats (Leonard et al., 2011; Leonard and Gulati, 2009). It was further found that the efficacy of IRL-1620 in a rat model of stroke was completely antagonized by BQ788 indicating an involvement of ETB receptors (Leonard et al., 2011; 2012).
Stroke and Cerebrovascular Disorders
Stroke is the rapid loss of brain function due to disturbance in the blood supply to the brain, which can be due to ischemia or a hemorrhage (Sims and Muyderman, 2009). It is the second leading cause of death and the fourth leading cause of disability worldwide (Mathers et al., 2009; Strong et al., 2007). It is also a predisposing factor for epilepsy, falls and depression (Fisher and Norrving, 2011) and is a foremost cause of functional impairments, with 20% of survivors requiring institutional care after 3 months and 15%-30% being permanently disabled (Steinwachs et al., 2000).
Stroke is divided into two broad categories: Ischemic strokes, caused by sudden occlusion of arteries supplying the brain, either due to a thrombus at the site of occlusion or formed in another part of the circulation. According to recent data released by the American Heart Association, 87% of strokes are classified as ischemic (Deb et al., 2010; Feigin et al., 2009; Roger et al., 2012). Hemorrhagic strokes, caused by bleeding from one of the brain's arteries into the brain tissue (subarachnoid hemorrhage) or arterial bleeding in the space between meninges (intra-cerebral hemorrhage).
The outcome after a stroke depends on the site and severity of brain injury. A very severe stroke can cause sudden death. Stroke affected area of the brain cannot function, which may result in an inability to move one or more limbs on one side of the body, inability to understand or formulate speech, or an inability to see one side of the visual field (Bath and Lees, 2000; Donnan et al., 2008). Early recognition of stroke is most important in order to expedite diagnostic tests and treatments.
Despite the severity of ischemic stroke, the only currently available FDA-approved pharmacological treatment is recombinant tissue plasminogen activator (rtPA), which dissolves the clot and restores blood flow to the brain. This treatment is complicated by the relatively short window of time between infarct and treatment (3-4 h) and the increased risk of subarachnoid hemorrhage (Micieli et al., 2009). A large number of other agents, broadly classified as neuroprotective and aiming to slow or stop the secondary damage associated with the ischemic cascade following stroke, have shown promise in the initial stages of research but have thus far failed to demonstrate efficacy in clinical trials (Ly et al., 2006). A new approach is therefore needed, one which has the potential to address both the restoration of blood flow and attenuate secondary damage to the penumbral area.
Following both ischemic stroke and subarachnoid hemorrhage, ET levels in the blood and ET immunoreactivity in the tissues are elevated (Asano et al., 1990; Rebello et al., 1995b; Viossat et al., 1993). A demonstration that the increase in ET levels coincides with a decrease in regional blood flow in the ischemic areas of the brain following experimental stroke led to the investigation of several ET antagonists in the treatment of focal ischemic stroke (Patel et al., 1995). Although some ETA specific and ETA/B non-specific antagonists have shown promise in experimental stroke models, others have not (Barone et al., 2000; Barone et al., 1995; Briyal and Gulati, 2010; Briyal et al., 2007; Briyal et al., 2012a; Gupta et al., 2005; Kaundal et al., 2012; Zhang et al., 2008; Zhang et al., 2005). Overall, this approach has not been useful. It has been demonstrated that ETB receptors, which increase VEGF and NGF in the brain, are overexpressed at the time of birth and their expression decreases with maturity of the brain (Briyal et al., 2012b). It appears that ETB receptors present in large number in the CNS play a key role in its development. This fundamental information demonstrates the possible involvement of ETB receptor in the brain development to generate neurovascular plasticity of the brain that has been damaged following cerebral ischemia. It was found that stimulation of ETB receptors with intravenous IRL-1620, a highly selective ETB agonist, resulted in a significant elevation in cerebral blood flow in normal rats (Leonard and Gulati, 2009). In addition, functional ETB receptors have been shown to enhance proliferation of neuronal progenitors and to protect against apoptosis in the dentate gyms, olfactory epithelium, and cortical neurons (Ehrenreich et al., 1999; Laziz et al., 2011; Lee et al., 2003; Yagami et al., 2005). The evidence that a deficiency in ETB receptors leads to a poorer outcome following cerebral ischemia (Chuquet et al., 2002) and complete deficiency or blockade of ETB receptors leads to exacerbation of ischemic brain damage (Ehrenreich et al., 1999) led to the investigation of the role of ETB receptors in a model of ischemic stroke. When a majority of research on ET and stroke thus far has focused on antagonizing ETA receptors selectively or non-selectively in order to prevent excessive vasoconstriction, the effect of selectively activating ETB receptors in a focal stroke model was examined (Leonard et al., 2011; 2012; Leonard and Gulati, 2013).
In clinical practice at present there are two basic treatments, preventive treatment using long term antiplatelet or anticoagulant agents to reduce the risk of stroke, or acute treatment by fibrinolytics. However, less than 2% of patients are able to receive fibrinolytics (Font et al., 2010). Extensive research is being conducted in search of neuroprotective agents for possible use in acute phase of stroke, and of agents that can be used for neurorepair in later stages of stroke.